Homo- and heteromeric complexes of KCNQ channel subunits are the molecular correlate of the M-current, a neuron-specific voltage-dependent K(+) current with a well established role in control of neural excitability. We investigated the effect of KCNQ channel modulators on the activity of dopaminergic neurons in vitro and in vivo in the rat ventral mesencephalon. The firing of dopaminergic neurons recorded in mesencephalic slices was robustly inhibited in a concentration-dependent manner by the KCNQ channel opener N-(2-amino-4-(4-fluorobenzylamino)-phenyl) carbamic acid ethyl ester (retigabine). The effect of retigabine persisted in the presence of tetrodotoxin and simultaneous blockade of GABA(A) receptors, small-conductance calcium-activated K(+) (SK) channels, and hyperpolarization-activated (I(h)) channels, and it was potently reversed by the KCNQ channel blocker 4-pyridinylmethyl-9(10H)-anthracenone (XE991), indicating a direct effect on KCNQ channels. Likewise, in vivo single unit recordings from dopaminergic neurons revealed a prominent reduction in spike activity after systemic administration of retigabine. Furthermore, retigabine inhibited dopamine synthesis and c-Fos expression in the striatum under basal conditions. Retigabine completely blocked the excitatory effect of dopamine D(2) autoreceptor antagonists. Again, the in vitro and in vivo effects of retigabine were completely reversed by preadministration of XE991. Dual immunocytochemistry revealed that KCNQ4 is the major KCNQ channel subunit expressed in all dopaminergic neurons in the mesolimbic and nigrostriatal pathways. Collectively, these observations indicate that retigabine negatively modulates dopaminergic neurotransmission, likely originating from stimulation of mesencephalic KCNQ4 channels.